Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

Abstract

SUMMARY Proper maintenance of mitochondrial activity is essential for metabolic homeostasis. Widespread phosphorylation of mitochondrial proteins may be an important element of this process; yet little is known about which enzymes control mitochondrial phosphorylation, or which phosphosites have functional impact. We investigate these issues by disrupting Ptc7p—a conserved but largely uncharacterized mitochondrial matrix PP2C-type phosphatase. Loss of Ptc7p causes respiratory growth defects concomitant with elevated phosphorylation of select matrix proteins. Among these, Δptc7 yeast exhibit an increase in phosphorylation of Cit1p—the canonical citrate synthase of the tricarboxylic acid cycle—that diminishes its activity. We find that phosphorylation of S462 can eliminate Cit1p enzymatic activity likely by disrupting its proper dimerization, and that Ptc7p-driven dephosphorylation rescues Cit1p activity. Collectively, our work connects Ptc7p to an essential TCA cycle function and to additional phosphorylation events that may affect mitochondrial activity inadvertently or in a regulatory manner.

A path to the powerhouse: systems‐to‐structure approaches for studying mitochondrial proteins

“…We have shown that Ptc6p is the yeast pyruvate dehydrogenase complex (PDC) phosphatase and that Ptc7p likely regulates multiple proteins, including citrate synthase (CS). After Grimsrud et al and Guo et al…”

“…By integrating these comparative phosphoproteomics with analyses of protein‐protein interactions via AE‐MS and in vitro biochemistry, we established Ptc6p as the S. cerevisiae pyruvate dehydrogenase complex phosphatase, thereby filling a longstanding gap in our understanding of yeast post‐translational metabolic regulation . We then connected Ptc5 and Ptc7 to distinct sets of ∼10–20 mitochondrial phosphoproteins each from amongst thousands of observed phosphoisoforms spanning diverse pathways . In particular, we demonstrated that Ptc7p‐driven dephosphorylation of mitochondrial citrate synthase rescues its ability to dimerize into an active form, and thus may be important for maintaining optimal mitochondrial activity .…”

“…To begin connecting these phosphorylation events to specific regulatory enzymes, we focused on a set of poorly characterized mitochondrial phosphatases—Ptc5, Ptc6, and Ptc7. Despite their high overall homology, knockout of each phosphatase in S. cerevisiae caused markedly different changes to the mitochondrial phosphoproteome . By integrating these comparative phosphoproteomics with analyses of protein‐protein interactions via AE‐MS and in vitro biochemistry, we established Ptc6p as the S. cerevisiae pyruvate dehydrogenase complex phosphatase, thereby filling a longstanding gap in our understanding of yeast post‐translational metabolic regulation .…”

“…Despite their high overall homology, knockout of each phosphatase in S. cerevisiae caused markedly different changes to the mitochondrial phosphoproteome. 29,30 By integrating these comparative phosphoproteomics with analyses of protein-protein interactions via AE-MS and in vitro biochemistry, we established Ptc6p as the S. . Phosphoproteomics define functions for orphan phosphatases.…”

Multi-omic Mitoprotease Profiling Defines a Role for Oct1p in Coenzyme Q Production

“…The acetone was aspirated, and the slides were allowed to air-dry (~2 min). The samples were blocked with BSA-PBS ([1% BSA in PBS], 2 mL, 30 min, ~23 °C), and incubated with primary antibodies (Sigma F1804, 1 mg/mL stock anti-FLAG primary Ab at a 1:2000 dilution in PBS-BSA; in-house made by biomatik anti-Cit1p antibody (Guo et al, 2017) at a 1:500 dilution in PBS-BSA; 1 mL, 12 h, 4 °C). The samples were washed 5 times with PBS-BSA (2 mL, ~23 °C ) and incubated with secondary antibodies diluted in PBS-BSA [1 µg/mL working concentration for each: Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate (Thermo A-11005) and Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Cat# A-11008)] (1 mL, 2 h, ~23 °C , in the dark).…”